Platelets play an important role in hemostasis by adhering to the surface of a damaged blood vessel.
However, it is known that platelet aggregation is primarily responsible for the formation of thrombus and that the formed thrombus obstructs a blood vessel. This obstruction prevents the adequate supply of oxygen and nutrients to tissues and organs and thereby causes ischemic diseases in circulatory organs as represented by myocardial infarction and cerebral infarction. At present, the mortality rates of these ischemic diseases follow that of cancer, which has become a significant social problem.
When medical treatments involving the extracorporeal circulation of blood, as exemplified by the use of an artificial heart and lung during surgical operations and renal dialysis for patients with renal failure, are conducted, blood coagulation may be caused by the activation and aggregation of platelets, which greatly disturb the performance of such treatments.
Thus, the prevention of thrombus formation and blood coagulation is an important matter in avoiding the occurrence of ischemic diseases or in safely conducting extracorporeal circulation.
Platelets are activated by the binding, to receptors on a platelet membrane, of thrombin present in plasma, connective tissue proteins such as collagen present in subendothelial tissues that may become exposed by damage to a blood vessel and other substances. Platelets are also activated by the binding of released adenosine diphosphate (ADP), adrenaline, serotonin, thromboxane (TX) A2 and the like to membrane receptors in a manner like autosecretion. Two kinds of glycoprotein units which compose a fibrinogen receptor are presented on the cell surface and associated to form a receptor complex (gpIIbIIIa), whereby aggregation via a fibrinogen bridge is induced.
Patients with thrombasthenia characterized by congenital absence of gpIIb and gpIIIa do not have a capability for platelet aggregation. Therefore, it is clear that the binding of the gpIIbIIIa complex to fibrinogen is essential to platelet aggregation (Rouslahti et al., Science, 238, 491 (1987)).
Attempts have been made to prevent thrombus formation by the inhibition of platelet aggregation utilizing the properties of the gpIIbIIIa complex. For example, Coller et al. reported that an F(ab').sub.2 fragment of a monoclonal antibody against the gpIIbIIIa complex has a strong inhibitory action on platelet aggregation and verified that a platelet aggregation-inhibiting agent could be developed utlizing this action (Blood, 68, 783, (1986)).
Although it is recognized that the monoclonal antibody has the potential as a therapeutic agent for inhibiting platelet aggregation, there is an apprehension for the possible production of antibodies against the monoclonal antibody by its repeated administration, since it is in itself a large protein.
Therefore, it has been desired to develop platelet aggregation-inhibiting agents containing as active ingredients non-immunogenic small compounds that have the properties of antagonists to the gpIIbIIIa complex.
Studies on the binding of fibrinogen to the gpIIbIIIa complex have been conducted aggressively. These studies started with the finding of arginine-glycine-aspartic acid (RGD) as an amino acid sequence common to cell adhesive molecule by a series of studies conducted by Ruoslahti et al. (Ruoslahti et al., Nature 309, 30-33 (1984)). The study of receptors recognizing the RGD sequence verified that the gpIIbIIIa complex is a receptor classified in an integrin family recognizing the RGD sequence (Phillips et al., Blood, 71, 831-843 (1988)) and that this complex especially recognizes two RGDF (-phenylalanine) sequences present in the fibrinogen molecule, thereby binding with the fibrinogen (Andrieux et al., J. Biol. Chem., 264, 9258-9265 (1989)).
Furthermore, it is known that the gpIIbIIIa complex binds to von Willebrand factor, fibronectin, vitronectin and thrombospondin which have the RGD sequence as well as fibrinogen (Pytela et al., Science., 231, 1559 (1986) and Cell, 42, 439 (1985)).
It is expected from these findings that synthetic peptides containing the RGD sequence inhibit the binding of the gpIIbIIIa complex to fibrinogen and thereby inhibit platelet aggregation. In fact, it was reported that 400 .mu.M of a synthetic peptide GRGDSP completely inhibited the aggregation of platelets activated by ADP (Plow et al., Proc. Natl. Acad. Sci. USA., 82, 8057-8061 (1985)). In addition, it has been verified that RGDS at concentrations of 46-50 .mu.M inhibits 80-90% of platelet aggregation in a concentration-dependent manner (Plow et al., Blood, 70, 110-115 (1987)). Moreover, it has been revealed that a peptide RGDF exhibits platelet aggregation-inhibiting activity 4-5 times as strong as RGDS (Harfinest et al., 71, 132-136 (1988)).
Japanese unexamined patent publication (hereinafter referred to as "KOKAI") Nos. Hei 1-190699 and Hei 2-62892, EPO 422937 A1 and U.S. Pat. No. 4,952,562 (hereinafter referred to as "USP") disclose tetrapeptide derivatives containing the RGD peptide. KOKAI No. Sho 63-215696 discloses derivatives consisting of peptides. KOKAI Nos. Hei 3-118331 and Hei 2-62892 and WO 91/01331 disclose derivatives having the cyclic structure of the RGD peptide.
It should be noted here that blood is usually transfused in separate components that are selected in accordance with use and whole blood transfusion is rarely conducted today. A platelet preparation for blood transfusion for use in such component transfusion is the blood preparation produced by a method of formulating the whole blood obtained by blood donation or that produced by component donation using the apheresis method. In usual practice, this platelet preparation is charged in a special preservative bag made of polyolefin or polyvinyl chloride immediately after the preparation treatment and stored at room temperature while stirring.
It has recently become clear that the adhesion of cells to extracellar matrix proteins is related to various diseases. In particular, it is being unrevealed that the adhesion is closely related to the mechanism of reoccurrence of tumor due to tumor metastasis.
The number of cases in which tumor can be removed by surgery is increasing due to improvements in the techniques of early detection and surgical operational techniques by the establishment of methods of tumor diagnosis. However, the mortality rate of tumor is increasing. The reoccurrence of tumor resulting from the metastasis of tumor cells is predominantly responsible for the high mortality rate. It may well be said that the reoccurrence of tumor can be prevented fairly effectively if a substance capable of suppressing the metastasis of tumor is found. Up to now, no effective agent in suppressing the metastasis of tumor has been developed.
Although the mechanism of the metastasis of tumor is not completely understood, it has been gradually revealed by recent studies. The metastasis of tumor has two very important steps, that is, the release of tumor cells from the primary lesion into blood and lymph and the transfer of tumor cells from lymph into tissues. Extracellular matrix proteins which compose the basement membrane of blood vessels are believed to provide a foothold for cell transfer.
In the basement membrane, various extracellular matrix proteins having the RGD sequence are present, such as fibronectin, collagen, vitronectin and laminin. The RGD sequence plays a very important role in the adhesion of tumor cells to the extracellular matrix proteins. Therefore, it has been pointed out that compounds inhibiting the adhesion of tumor cell to the basement membrane of blood vessels, particularly RGD analogues can inhibit tumor cells from releasing into blood and transfering from the inside of a blood vessel to the outside thereof and thereby suppress the metastasis of tumor.
In recent years, in order to develop highly active agents having high in vivo stability, there have been aggressively conducted studies in which compounds having a structure which does not naturally occur are derived from RGD peptide as a key compound (Hartman et al., J. Med. Chem., 35, 4640-4642 (1992) and Callahan et al., ibid, 35, 3970-3972 (1992)). These compounds are useful as platelet aggregation-inhibiting agents for oral administration which are susceptible to the action of protease; however, they are expected to manifest toxicity (this problem often occurs in the derivation to non-natural structures) and to have such a side effect that the drugs are not metabolized but accumulated in the human body. Hence, there exists a strong concern for safety.
An improvement in the in vivo stability of compounds leads to the persistence of platelet aggregation-inhibiting action and blood coagulation-inhibiting action, thereby potentially inhibiting for a long period of time the important physiological actions inherently possessed by platelets, as exemplified by the inducement of hemorrhagic tendency and the like.
In particular, at the time of extracorporeal circulation or surgical operation, the persistence of platelet aggregation-inhibiting action and blood coagulation-inhibiting action becomes a problem. For example, it has been reported that even heparin that is a drug from an organism and which is actually administered to suppress blood coagulation has such a significant side effect that it acts beyond an appropriate period and thereby hemorrhagic tendency is induced (Tadao Akizawa, et al., NIHON RINSHO, vol. 43, 377-391 (1985)).
Accordingly, in the case where a platelet aggregation-inhibiting agent is deliberately used to inhibit temporarily the aggregation ability of platelets at the time of extracorporeal circulation or surgical operation, it is not only desired that it has an excellent platelet aggregation-inhibiting ability but also required that it has good safety characteristics in that it acts for a reasonable period of time while it is rapidly metabolized after inactivation to compounds having no side effects. As described above, the RGD peptides per se do not have such high platelet aggregation-inhibiting and blood coagulation-inhibiting actions as to warrant use in clinical practice. However, the RGD peptides have an excellent characteristic in that they are broken down by protease inherently present in an organism to amino acids which are safe and useful to the organism.
The inventors utilized this characteristic and produced highly active peptide derivatives that have various purposive working times in organisms, that have excellent platelet aggregation-inhibiting ability and blood coagulation-inhibiting ability and which have a structure that is as homologous as possible to naturally occurring peptides. An object of the present invention is to provide platelet aggregation-inhibiting agents comprising the peptides as active ingredients. A further object of the present invention is to provide blood coagulation-inhibiting agents having reduced side effects which are useful at the time of extracorporeal circulation and surgical operations.
At present, the platelet function (platelet aggregation ability) of the above described platelet preparations for blood transfusion is significantly reduced during storage or preservation, which is one of the factors that prevent useful blood transfusion. Since there is no method available today for successfully storing the platelet preparations while avoiding the reduction of the platelet function during storage, the establishment of an effective method for storing the platelet preparations is being studied in the world. Therefore, a still further object of the present invention is to provide agents for protecting platelet preparations for blood transfusion that comprise the peptides as active ingredients.
It is believed that the reduction of platelet function during storage is caused mainly by 1 the activation and aggregation of platelets, which are produced by various physical stimulations that occur at the time of collecting blood, treating and storing preparations and 2 the lowering of the pH of preservative solutions. Although improvements in preservative solutions and preservative systems for the purpose of pH control have been aggressively made in recent years, it can not be said that they are sufficiently effective at present.
In addition, on the basis of the idea that the reduction of platelet function during storage may be suppressed by inhibiting the aggregation of platelets, several trials of adding platelet aggregation-inhibiting substances such as aspirin, prostaglandin and the like to store blood have been made at an experimental level. However, these compounds have a fatal disadvantage in that they are not easily broken down in vivo and therefore, if platelet preparations comprising these compounds are transfused, the condition in which systemic blood is hard to coagulate is maintained for at least several hours. Therefore, they are not used in clinical practice.
The inventors believed that the reduction of the aggregation ability of platelets and the decrease in the number of platelets can be prevented by adding the aforementioned highly biodegradable and safe derivative peptides having a strong platelet aggregation-inhibiting activity to platelet preparations for blood transfusion. Thus, an additional object of the present invention is to develop compounds having the ability to protect platelets in preparation packs during storage.
Moreover, other objects of the present invention are to provide cell adhesion-inhibiting agents and tumor metastasis-inhibiting agents which comprise the above mentioned peptides as active ingredients, utilizing the cell adhesion-inhibiting action of the peptides of the present invention.